Project Abstract An immune response begins with the detection of infection by pattern recognition receptors (PRRs). Notably, some cytosolic PRRs allow for the nucleation and sequential oligomerization of large signaling complexes called the inflammasome. The inflammasome activates the protease caspase-1. Caspase-1 cleaves pro-form IL-1 family members that exist in a latent state in the cytosol of primed cells. Caspase-1 (and caspase-11) also cleave the latent cytosolic protein gasdermin D (GSDMD) that when cleaved becomes the executioner of the inflammatory cell death program termed pyroptosis by incorporation of N terminal fragments into the inner leaflet of the plasma membrane. GSDMD pore formation is thought to ultimately lead to cellular lysis. IL-1 family members are unable to be secreted by the conventional protein secretion pathway involving trafficking through the biosynthetic pathway. Instead, it is thought that inflammasome activation leads to lytic cell death that allows for the release of cytosolic contents, including cleaved IL-1 family members, into the extracellular space through large membrane tears. A new cell fate that also involves the inflammasome and activation of caspase-1 has been termed phagocyte hyperactivation. The hyperactive cell fate is characterized by the ability of non-pyroptotic (living) cells to release cytokines from the conventional biosynthetic pathway while also releasing cytosolic inflammatory mediators such as IL-1 family members. A major question remains based on our current understanding of inflammasome signaling and IL-1? release: how can cells with active inflammasomes remain viable and also release cytosolic IL-1? The goal of this project is to determine how the pyroptotic regulator GSDMD mediates a non-pyroptotic role in unconventional protein secretion. To accomplish this goal, we aim to determine if intrinsic differences exist between pyroptotic and hyperactive inflammasomes that differentially regulate the extent of GSDMD pore formation in lytic (pyroptotic) and non-lytic (hyperactive) contexts. We also aim to determine the location of cleaved GSDMD fragments after inflammasome signaling and perturbation of membrane repair to determine the ultimate fate of these GSDMD pores in living cells. Delineation of this novel mechanism of IL-1 activation and release that does not require cell death may lead to the design of more efficacious vaccine adjuvants and shift our understanding of the impact of inflammasome signaling on host immunity and defense against pathogens.